The gram-positive pathogen Streptococcus agalactiae (group B streptococci, GBS) is the principal cause of human neonatal pneumonia, sepsis and meningitis. GBS is also an emerging pathogen of immunocompromised adults. We recently identified and characterized a novel eukaryotic-type serine/threonine protein kinase (Stk1) and its cognate phosphatase (Stp1) in GBS. Mutants of this signal transduction pathway exhibited pleiotropic effects on cell growth, virulence and segregation of GBS, indicating the importance of this pathway in the regulation of various cellular processes. In vitro phosphorylation studies revealed that these enzymes are essential for reversible phosphorylation of many GBS proteins. Using mass spectrometric analysis, we identified one of these targets as a anganese-dependent inorganic pyrophosphatase (PpaC). Pyrophophatases are critical for regulation of biosynthetic reactions in the cell. Based on our results, we hypothesize that this signal transduction pathway and post-translational modification of its targets are crucial for normal cellular functions in GBS. A combination of molecular, biochemical and proteomic approaches will be used to elucidate the role of this signal transduction pathway and its physiological substrates in growth and virulence of GBS. In aim 1, we will identify and characterize the upstream and downstream targets of this pathway. We will utilize modem proteomic techniques such as liquid chromatography and mass spectrometry to identify other key targets of this signal transduction pathway. We will perform deletion analysis and protein cross-linking studies, to identify proteins that bind to and activate Stk1. In Aim 2, we will complete functional characterization of the identified physiological substrate of this signal transduction pathway, PpaC. In Aim 3, we will construct mutations in ppaC and a few other regulated targets of this pathway, identified in aim 1, to assess their role in growth and survival of GBS. We anticipate that some of these genes including ppaC will be essential for GBS growth. As mutants inessential genes are not viable, we will use the modem RNA interference technology to evaluate their role in GBS growth and survival. Collectively, these studies will determine the biological significance of this signal transduction pathway and lead to the identification of novel targets of GBS, which may provide insights into their potential as antimicrobial targets.